Proteins and nucleic acids have demonstrated great value as research tools and as human therapeutics. Due to the inability of most macromolecules to spontaneously enter cells, however, exogenous proteins and nucleic acids are largely restricted to interacting with extracellular targets even though most targets of potential biological and medical interest are thought to be intracellular. Methods to deliver proteins and nucleic acids into mammalian cells are therefore of considerable interest to the biomedical community because they may address the major outstanding challenge facing the use of proteins and nucleic acids as intracellular probes and therapeutic agents. As part of an effort to study the problem of protein aggregation, we recently mutated virtually all non-conserved surface exposed residues of several different proteins to Lys or Arg, creating superpositively charged proteins, or to Asp or Glu, creating supernegatively charged proteins. We discovered that some of the resulting supercharged proteins can retain their native folding and function, but are virtually immune to aggregation. Remarkably, the superpositively charged proteins we generated, including a variant of green fluorescent protein with a net theoretical charge of +36, very potently enter diverse types of mammalian cells, including several cell lines resistant to traditional transfection methods. When pre-mixed with siRNA or plasmid DNA, these supercharged GFPs were able to deliver nucleic acids very efficiently into all five cell lines tested. In four of the five cell lines, siRNA-based gene silencing or plasmid-based gene expression was also observed. Our very recent preliminary results suggest that supercharged proteins can also deliver proteins of interest into mammalian cells, and that supercharged proteins can also deliver nucleic acids and functional proteins in vivo. These findings suggest that supercharged proteins represent a promising new class of macromolecule delivery agents that merit further study as tools for perturbing cells and as a potential component of future macromolecular therapeutics. Here we propose to study the potential of supercharged proteins to address key problems in macromolecule delivery. Specifically, we seek to develop supercharged proteins as effective tools for delivering proteins into mammalian cells, to elucidate and improve the molecular properties of supercharged proteins that confer their potent macromolecule delivery activities including the ability to escape from endosomes, to discover naturally occurring superpositively charged human proteins that may serve as promising starting points for the development of non-immunogenic delivery systems, and to begin the first studies using supercharged proteins to deliver functional proteins or nucleic acids, including those that are therapeutically relevant, into live animals. PUBLIC HEALTH RELEVANCE: Methods to deliver proteins and nucleic acids into mammalian cells are of considerable interest to the biomedical community because they are thought to address the major outstanding challenge facing the use of proteins and nucleic acids as intracellular probes and therapeutic agents. We propose to study and develop the ability of a new class of proteins that we recently discovered, supercharged proteins, to be used to deliver into cells externally applied DNA, RNA, and proteins both in the test tube and in living animals. The resulting methods and insights may represent a significant step towards the widespread use of proteins and nucleic acids as drugs to address deficiencies and biological targets inside cells.